Callicarpa stoloniformis (Lamiaceae), a new species from Southeast China based on morphological characters and phylogenetic evidence

Abstract Callicarpa stoloniformis sp. nov. (Lamiaceae) is described as a new species from Fujian Province of China on the basis of both morphological and molecular data. The new species is morphologically most close to C. hainanensis. However, it can be distinguished from the latter by its unique procumbent life form, adventitious roots at nodes, papery leaves, cup‐shaped or campanulate calyx, truncate or shallow fissure calyx lobes, and smaller fruits. In addition, the new species is also similar with C. basitruncata, a species only known from the original description and the photograph of holotype, but it can differ from the latter by its procumbent shrub, purple terete branchlets with apparent linear lenticels, adventitious roots at nodes, and papery larger leaves with prominently cordate leaf base. Original photographs, illustration, distribution map, and a comparative morphological table, as well as an identification key of the related taxa are provided.


| INTRODUC TI ON
a single taxon, C. peichieniana (1982: 78), which was characterized by strongly curved stem hair; while other species with various hair types were assigned to Subgen. Callicarpa. In recent years, evidences from morphology, palynology, and molecular phylogeny have proved that there was a sister relationship between Callicarpa and subfamily Prostantheroideae endemic in Australia and both formed a basal clade in Lamiaceae (Cai et al., 2021;Li et al., 2016;Ma, 2013;Ma & Zhang, 2012;Zhao et al., 2021).
Recently, more comprehensive phylogenic studies on the framework of Lamiaceae suggested establishing a new monotypic subfamily Callicarpoideae (Zhao et al., 2021). Liu et al. (2022) combined the analysis of phylogeny, biogeography, and statistics, and revealed that the fruit color of Callicarpa was strongly associated with geographical distribution. Besides, they also suggested all fruit colors of the genus were involved in dispersal events, and inferred violet fruit promoted diversification in Callicarpa and drove the evolution and diversity of different fruit colors between regions. In Asia, a number of Callicarpa are valued traditional medicinal plants and several of them, such as C. formosana (Trimen et al. 1882: 358), C. macrophylla (Vahl & Forssk, 1794: 13), C. kwangtungensis (Chun, 1934: 302), and C. nudiflora (Hooker et al. 1841: 206) have been intensively studied and included in Chinese Pharmacopoeia (2020 edition). Furthermore, drug products of them are widely used in clinical (eg. Luo Hua Zi Zhu Pian, Kang Gong Yan Pian, Fu Yan Ning Jiao Nang, Wu et al., 2018).
As part of our ongoing taxonomic revision of Callicarpa in China, some interesting specimens were collected from Neikengkou, Nanjing County (Fujian Province, China). After examination of the floras or monographs from China and the adjacent Asian regions (Bramley, 2009(Bramley, , 2013Chen & Gilbert, 1994;Fang, 1982;Leeratiwong et al., 2009;Ma, 2013), as well as analysis of herbarium specimens, our discovered plants cannot be placed in none of the current known species of Callicarpa. Based on morphological and molecular phylogenetic data, we decided to propose the newly collected plants as a new species.

| Morphology
Field surveys of the putative new species were carried out in Neikengkou, Nanjing County, Zhangzhou City, Fujian Province, during the period of June-December 2021. Syndromes of the habitat, life form, hairs, leaves, cyme, and fruit were surveyed with naked eyes or a hand magnifier and photographs were taken in the field.
Detailed characters of glandular, hair, floral anatomy, ovary, and seed were observed with stereomicroscope (NIKON SMZ25) in the laboratory. Examination of specimens was also made for morphological comparison with the related species (herbaria visited: GAUA, IBSC, PE, CSH, IBK, NY, G, E, A, P, TI, and K; acronyms according to Thiers, 2022). The size of leaves, cymes, flowers, and fruits was measured with a vernier caliper in the laboratory. All voucher photos of the putative new species were deposited in the "Chinese Field Herbarium" (https://www.cfh.ac.cn/album/ ShowS pAlbum. aspx?spid=94377).

| Molecular phylogeny
A total of 14 species representing the main sections, subsections, and series of Chinese Callicarpa were selected for molecular phylogenetic analyses with two species from Prostantheroideae: Dasymalla teckiana (Conn et al. 2011: 6) and Dicrastylis parvifolia (Mueller, 1861: 160) as the outgroups. Total DNA was extracted from fresh leaves dried with silica gel using a modified CTAB method (Doyle & Doyle, 1987). Two nuclear [internal transcribed spacer (ITS) and external transcribed spacer (ETS)] and five chloroplast (matK, rpl32-trnL, trnH-psbA, psbJ-petA, and trnS-trnG intergenic spacer) regions were chosen for phylogenetic analyses. PCR amplification, sequencing, sequence editing, and sequence assembly were performed according to Katoh and Standley (2013) and Kumar et al. (2016). The primer pairs used for PCR were shown in Table 1. Detailed information on the DNA regions and GenBank accession numbers of the species investigated in this study were shown in Table 2.
The bootstrap (BS) percentage for each branch was estimated by running 1000 bootstrap replicates. For BI analysis, ModelFinder (Kalyaanamoorthy et al., 2017) was used for the selection of the most appropriate evolutionary model (nucleotide substitution model) (Edge-linked) using BIC criterion. The best partition models with BIC criterion were as follows: for ETS and ITS, HKY + F + G4; for matK, psbJ-petA, rpl32-trnL and trnG-trnS intergenic spacer, GTR + F + G4; for trnH-psbA intergenic spacer, F81 + F. The run with 1,000,000 generations was conducted. Four Markov chains with two runs were implemented and sampled every 100 generations, and the first 25% of all trees were regarded as "burn-in". The majority consensus of the remaining trees was generated to show posterior probability (PP) support for clades. Stationarity was determined in Tracer v1.7.1 (Rambaut et al., 2018) and was considered to be attained when ESS > 200 or when the average standard deviation of the split frequencies was <0.01.

| Morphology
The putative new species is similar to Callicarpa hainanensis Ma & Zhang (2012: 573) and C. basitruncata Merrill ex Moldenke (1951: 406), and their detailed morphological comparison from life form, stem, leaves, cymes, calyx, corolla, and fruit were list in Table 3. The new species is most similar to C. hainanensis, and both share lots of common characteristics in branchlets, indumentum, leaf base, filament length, and the way of anther opening. Even so, it can be distinguished from C. hainanensis by its unique life form, adventitious roots at nodes, the morphology of calyx, calyx lobes, and the size of fruits ( cymes, and calyx, but it can differ from the latter by its procumbent life form, terete branchlets with adventitious roots at nodes, apparent linear lenticels, and leaf morphology ( Table 3, Figures 1-3). In terms of leaf blade base, the putative new species is slightly similar to C. rubella and C. longipes, which all have cordate leaf bases.
Obviously, it has filaments slightly shorter or as long as corolla, with larger oblong anthers, while the latter two species all have filaments twice or more than twice as long as the corolla, with small, ovate anthers.

| Molecular phylogeny
The combined analysis indicated that Callicarpa is monophyletic (PP = 1.00, BP = 100%, Figure 4) with respect to the groups considered. Two main clades are formed and our phylogenetic result seems not being consistent with Chang's (1951) traditional classification system based on the length of filament, and morphology of anther, which is widely recognized (Bramley, 2009(Bramley, , 2013Fang, 1982). The putative new species forms a well-supported sister clade with the clade composed by members of section Eucallicarpa: C. rubella and C. longipes (PP = 1.00, BP = 98%), and then, these three species form a weakly supported sister clade with C. japonica (section Verticirima) (PP = 0.63, BP = 48%, Figure 4). Although it forms a sister clade with C. rubella and C. longipes and shares few common characteristics in morphology, such as cordate leaf base, it can be obviously distinguished from the latter two species by its unique procumbent life form, adventitious roots at nodes, and shorter filaments or as long as corolla (typical characters of section Verticirima). Moreover, it also can be easily identified from C. japonica by its procumbent life form, adventitious roots at nodes, stellate hairs on the young branchlets and inflorescence, and cordate leaf base. It is strange that C. hainanensis which we think the most morphologically similar species with C. stoloniformis (mentioned above), firstly forms a clade with C. brevipes (Benth.) Hance (1886: 233) and C. giraldii Hesse ex Rehd. (Rehder, 1914: 629), subsequently form a moderately supported sister clade with the clade including C. stoloniformis, C. japonica, C. rubella, and C.